Anti-Collagen II antibody (ab34712)

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Immunostaining was performed using standard protocols according to the Abcam/Santa Cruz official website (https://www.abcam.cn/protocols/ihc-tissue-processing-protocol and https://www.scbt.com/scbt/zh/resources/protocols/immunofluorescence-cell-staining). Briefly, sections were dewaxed and hydrated and then boiled in EDTA antigen repair solution (pH 8.0) for 20 min for antigen retrieval. Tissues were treated with 3% H2O2 to block endogenous peroxidase. The sections were then incubated with normal goat serum for blocking and subsequently with primary antibodies including mouse collagen II (Col II; Abcam, 1:200, ab34712), osterix (OSX; Santa Cruz, 1:200, sc-393,060), osteopontin (OPN; Santa Cruz, 1:200, sc-73,631), and vascular endothelial growth factor (VEGF; Santa Cruz, 1:200, sc-7269) at 4 °C overnight. Negative control slices were incubated in PBS without antibodies. For immunohistochemical staining, the rest of the procedures were manipulated according to the PV-6001 Two-Step IHC Detection Reagent instructions. After coloration with 3,3’-diaminobenzidine (DAB) solution (ZSGB-BIO Corporation, China), the sections were counterstained with hematoxylin and the yellow or brown color was considered as positive staining. For the immunofluorescent assay, the slides were incubated with the secondary antibody conjugated with fluorescence-Alexa Fluor® 555 (CST Corporation, USA, #4409) for 1 h avoiding light, followed by counterstaining with 4’, 6-diamidino-2-phenylindole (DAPI), and then detected under fluorescence microscopy (Olympus DP80, Japan). Positive staining in the disc was quantified using ImageJ Pro Plus software (Media Cybernetics, Baltimore, MD, USA).

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